This invention relates generally to the amplification of DNA sequences by polymerase chain reaction (PCR) and, more particularly, to the alteration of DNA sequences using PCR.
Recombinant DNA techniques have revolutionized molecular biology and genetics by permitting the isolation and characterization of specific DNA fragments. Of major impact has been the exponential amplification of small amounts of DNA by a technique known as the polymerase chain reaction (PCR). The sensitivity, speed and versatility of PCR makes this technique amenable to a wide variety of applications such as medical diagnostics, human genetics, forensic science and other disciplines of the biological sciences.
PCR is based on the enzymatic amplification of a DNA sequence that is flanked by two oligonucleotide primers which hybridize to opposite strands of the target sequence. The primers are oriented in opposite directions with their 3' ends pointing towards each other. Repeated cycles of heat denaturation of the template, annealing of the primers to their complementary sequences and extension of the annealed primers with a DNA polymerase result in the amplification of the segment defined by the 5' ends of the PCR primers. Since the extension product of each primer can serve as a template for the other primer, each cycle results in the exponential accumulation of the specific target fragment, up to several millionfold in a few hours. The method can be used with a complex template such as genomic DNA and can amplify a single-copy gene contained therein. It is also capable of amplifying a single molecule of target DNA in a complex mixture of RNAs or DNAs and can, under some conditions, produce fragments up to ten kb long. The PCR technology is the subject matter of U.S. Pat. Nos. 4,683,195, 4,800,159, 4,754,065, and 4,683,202 all of which are incorporated herein by reference.
In addition to the use of PCR for amplifying target sequences, this method has also been used to generate site-specific mutations in known sequences. Mutations are created by introducing mismatches into the oligonucleotide primers used in the PCR amplification. The oligonucleotides, with their mutant sequences, are then incorporated at both ends of the linear PCR product. In addition to their mutated sequences, the primers often contain restriction enzyme recognition sequences which are used for subcloning the mutated linear DNAs into vectors in place of the wild type sequences. Although this procedure is relatively simple to perform, its applications are limited because appropriate restriction sites are not always conveniently located for substituting the mutant sequence with the wild-type sequence. Restriction sites can be incorporated into the wild-type sequences for subcloning. However, such extraneous sequences can cause detrimental effects to the function of the gene or resulting gene product. Moreover, PCR products typically contain heterogeneous termini resulting from the addition of extra nucleotides and/or incomplete extension of the primer-templates. Such termini are extremely difficult to ligate and therefore result in a low subcloning efficiency.
Several modifications of the PCR-based site-directed mutagenesis strategies have been developed to circumvent such limitations, but they too have undesirable features. The most prominent undesirable feature exhibited by these alternative methods is a low frequency of correct mutations. For example, inverse PCR (IPCR) is a method which amplifies a circular plasmid rather than a linear molecule, Hemsley et al., Nuc. Acid. Res. 17:6545-6551 (1989), which is incorporated herein by reference. In this technique, two primers which are located back to back on opposing DNA strands of a plasmid drive the PCR reaction. The resultant PCR product, a linear DNA molecule identical in length to the starting plasmid, contains any mutations which were designed into the primers. The product is then enzymatically prepared for ligation by blunting and phosphorylating the termini. Enzymatic treatment of the termini is a necessary step for ligation due to heterogeneous termini associated with PCR products. These treatments are likely to be incomplete and cause unwanted mutations as well as result in a low ligation and transformation efficiency due to the additional required steps.
Recombinant circle PCR (RCPCR), Jones and Howard, BioTechniques 8:178-183 (1990), and recombination PCR (RPCR), Jones and Howard, BioTechniques 10:62-65 (1991), on the other hand, are two methods similar to IPCR which do not require any enzymatic treatment. In RCPCR, two separate PCR reactions, requiring a total of four primers, are needed to generate the mutated product. The separate amplification reactions are primed at different locations on the same template to generate products that when combined, denatured and cross-annealed, form double-stranded DNA with complementary single-strand ends. The complementary ends anneal to form DNA circles suitable for transformation into E. coli.
RPCR is a technique that uses PCR primers having a twelve base exact match at their 5' ends, resulting in a PCR product with homologous double-stranded termini. Transformation of the linear product into recombination-positive (recA-positive) cells produces a circular plasmid through in vivo recombination. Although this method reduces the number of steps and primers used compared to RCPCR, the transformation and recombination of linear molecules is an inefficient process resulting in a correspondingly low mutation frequency.
Thus, a need exists for a PCR-based method which allows the rapid and efficient alteration of nucleotide sequences. The present invention satisfies this need and provides related advantages as well.